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Flow type staining kit and preparation method and application method thereof

A dyeing reagent and flow technology, applied in the field of intracellular protein molecule detection, can solve the problem of thimerosal toxicity and other problems, and achieve the effects of reducing non-specific staining, rich detection parameters and easy operation.

Pending Publication Date: 2022-01-04
苏州东岭生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Like sodium azide, thimerosal is also very toxic

Method used

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  • Flow type staining kit and preparation method and application method thereof
  • Flow type staining kit and preparation method and application method thereof
  • Flow type staining kit and preparation method and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Intracellular staining of Th1 / Th2 cells

[0067] Because in the quiescent state, the unactivated lymphocytes basically do not express or only express a small amount of cytokines, so it is difficult to detect. Only when it is activated, the synthesis of intracellular cytokines can increase. Therefore, the detection of intracellular cytokines must be activated by activators. The level of cytokines in the cells is difficult, so it is necessary to use blocking agents to prevent the secretion of cytokines to the outside of the cells for detection. Therefore, we activate T cells by adding PMA (phorbol ester) and Inomycin (ionomycin); then use monensin (BD company's trade name GolgiStop) to inhibit the secretion of intracellular cytokines to the outside of the cell, so that cytokines After synthesis, it accumulates in the cells, which is convenient for more effective detection of the secretion level of intracellular protein factors. The flow staining kit of the present invent...

Embodiment 2

[0078] Intracellular staining of 293t-ifng stably transfected cell line

[0079] Apply the flow staining kit of the present invention to prepare the cell suspension and apply it to the detection of proteins in the cytoplasm of the cells (starting from step 4). The specific operation steps are as follows:

[0080] Step 4.1: Cell fixation: Add 200ul Fix / Perm Buffer (1X) to each tube of cells, fully resuspend the cells, and incubate at 4°C in the dark for 12 minutes;

[0081] Step 4.2: Add 500ul 1X C-Perm / Wash Buffer to each tube of cells, centrifuge at 600g×5min, 4 ℃, discard the supernatant;

[0082] Step 4.3: Antibody incubation: Add 100ul 1X C-Perm / Wash Buffer and an appropriate volume of IFNg intracellular antibody to each tube, fully resuspend the cells, and incubate at 2-8°C for more than 1 hour or overnight in the dark;

[0083] Step 4.4: Add 500ul 1X C-Perm / Wash Buffer to each tube of cells, wash away the antibody, centrifuge at 600g×5min, 4 ℃, discard the supernatant; ...

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Abstract

The invention discloses a flow type staining kit and a preparation method and an application method thereof, and particularly relates to a preparation method and an application method of a flow type staining kit for detecting various protein molecules in mammal nucleated cell cytoplasm, and the kit comprises a flow type intracellular fixation / membrane rupture solution and a flow type intracellular membrane rupture / washing solution. The immobilization / membrane rupture liquid is composed of paraformaldehyde, saponin and a phosphate buffer solution; the membrane rupture / washing liquid consists of saponin, fetal calf serum, a metal ion chelating agent, a phosphate buffer solution and a preservative. The kit can be used in combination with various fluorescein-labeled antibodies, and cell membranes are perforated through membrane rupture / washing liquid, so that fluorescence-labeled antibody molecules enter cytoplasm to be combined with specific antigen proteins. And the cells are detected through a flow cytometer to obtain information of protein molecule expression in specific cytoplasm. The invention can be used for flow immunodetection of various cells.

Description

technical field [0001] The invention relates to an intracellular protein staining kit for flow analysis, a configuration method and an application method thereof, and belongs to the technical field of intracellular protein molecular detection. Background technique [0002] Flow cytometry (Flow Cytometry, FCM) is a technology that can perform one-by-one, multi-parameter, rapid qualitative, quantitative or sorting of biological particles such as single cells, microorganisms or synthetic microspheres in a fast linear flow state . Flow cytometry can achieve high-throughput detection, the number of cells detected per second can reach thousands, and the number of cells detected can reach up to millions; it can detect single cells or biological particles one by one; through multi-parameter , Multi-color fluorescence analysis to realize accurate identification and counting of cells, qualitative and quantitative analysis of cells, and sorting of biological particles such as cells ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533
CPCG01N33/533
Inventor 邓猛猛孟丽君
Owner 苏州东岭生物技术有限公司
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